Molecular biology, isotopic labeling, and refolding of membrane proteins in phospholipid bilayers.

Transcription

1 Molecular biology, isotopic labeling, and refolding of membrane proteins in phospholipid bilayers. 1

2 Preparation of membrane proteins for NMR experiments. Established bacterial overexpression systems. Over forty different constructs of various membrane proteins. Improved purification and refolding protocols. Efficient detergent removal and sample quality control by lipid analysis. Implemented various lipid bilayer systems. Proteoliposomes. Nanodiscs and macrodiscs. Bicelles. Implemented various isotopic labeling schemes. Several types of sparse labeling. Complementary selective labeling. Perdeuteration. Preparation of membrane proteins for NMR experiments plays an important role in our BTRC program together with the development in NMR probes, pulse programs, and structure calculations. 2

3 General protocol for sample preparation of membrane proteins. Vpu TM CXCR1 CXCR1 Vpu TM Lipid analysis by Evaporative Light Scattering Detection (ELSD) Before reconstitution After reconstitution The protocol can be applied to a wide range of membrane proteins from a small single transmembrane protein to a large seven transmembrane helical protein. retention (ml) 3

4 Expression, purification, and refolding of human GPCRs. Function & related diseases Fusion protein Expression vector & host cells Reconstitution lipids Yield (mg/l culture) CXCR1 Chemokine receptor Inflammatory responses GST pgex-2t BL21 DMPC DMPC/POPC 8 AVPR2 Arginine vasopression receptor Nephrogenic diabetes insipidus KSI pet-31b(+) C43(DE3) DMPC DMPC/POPC 2 b2ar Adrenergic receptor Asthma, obesity, type 2 diabetes KSI pet-31b(+) BL21(DE3)plysS DPPC/CHS 3 CB2* Cannabinoid receptor Immune system, Neurodegenerative disorders GST pgex-2t BL21 codon plus DMPC ~1 *Collaboration with Sean Xie s group at the University of Pittsburgh. The refolding and reconstitution protocol originally developed for CXCR1 was successfully applied to other GPCRs with minor modifications, and the multi-milligram quantities of the functional receptors were obtained. 4

5 Refolded GPCRs have biological activities. G-protein activation assay NMR binding experiments CXCR1 unbound EC 50 = 1 nm CXCR1 bound 1 H shift (ppm) b2ar Fluorescent antagonist binding Competitive CB2 in ligand DMPC binding displacement H 3 CP55940 assay CB H CP55,940 %specific binding 50 0 K i = 4.74 nm log[sr144528] M Xie and coworkers 5

6 Two-dimensional 13 C/ 13 C correlation MAS solid-state NMR spectra of three constructs of Vpu from HIV-1. Vpu Full Vpu Cyto Vpu TM QPIQIAIVALVVAIIIAIVVWSIVIIEYRKILRQRKIDRLIDRLIERAEDSGNESEGEISALVELGVELGHHAPWDVDDL EYRKILRQRKIDRLIDRLIERAEDSGNESEGEISALVELGVELGHHAPWDVDDL QPIQIAIVALVVAIIIAIVVWSIVIIEGRGGKKKK 81 Vpu Full Vpu Cyto Vpu TM ~ 2 mg of uniformly 13 C/ 15 N-labeled protein, lipid/protein = 5 10 (w/w) 6

7 Membrane environment affects the structure of p7 from hepatitis C virus. P7 ALENLVVLNAASVAGAHGILSFLVFFSAAWYIKGRLAPGAAYAFYGVWPLLLLLLALPPRAYA 63 DHPC detergent micelles DMPC lipid bilayers C N C In general, membrane proteins are more stable in lipid bilayers than in detergent micelles, suggesting that the p7 structure in lipid bilayers is more relevant to the native structure in cell membranes. N 7

8 * * * * Terminal truncation affects the structure of the bacterial mercury transporter MerF. MerF MerFt * * KDPKTLLRVSIIGTTLVALSSFTPVLVILLGVVGLSALTGYLDYVLLPALAIFIGLTIYAIQRKRQADASSTPKFNGVKKS IGTTLVALSSFTPVLVILLGVVGLSALTGYLDYVLLPALAIFIGLTIYAIQRKRQADASS 81 A52 A19 MerF MerFt A N C N C It is important to study the full-length membrane proteins in lipid bilayers in order to obtain accurate structurefunction relationships of membrane proteins. 8

9 Mobile N-terminal region of CXCR1 is immobilized upon interaction with its ligand interleukin-8. Unbound 1TM-CXCR1 IL-8 bound 1TM-CXCR1 unlabeled IL-8 15 N-1TM-CXCR1 15 N-1TM-CXCR1 15 N shift (ppm) 15 N shift (ppm) We have determined the structure of the unmodified full-length chemokine receptor CXCR1 in lipid bilayers. And now we continue working on the structure of the complex of the receptor, ligand and G-proteins in order to understand the activation mechanism of CXCR1. 9

10 Cell-free expression of membrane proteins. Quick production of membrane proteins. Selective amino acid labeling. Unnatural amino acid incorporation. Cell-free Cell-based TM-CXCR MSP GST TM-CXCR1 1TM-CXCR1 lipids MSP Nanodisc 1. Supernatant in the presence of nanodiscs 2. Precipitate in the absence of nanodiscs 3. 1TM-CXCR1 and GST in the presence of detergents MembraneMax expression kit: 10

11 Unnatural amino acid incorporation into membrane proteins. HQA 1 : (2-Amino-3-(8-hydroxyquinolin-3-yl)propanoic acid. Forms stable complexes with metal ions and lanthanides. Apply to distance measurement by Paramagnetic Relaxation Enhancement. pevol-aars 2 prok term trna prok prom PstI (4801) Xho I (5336) ApaLI (5112) glns T aars CmR pevol 6124 bp p15a arac Nde I (3874) glns' rrnb SalI (3453) aars arabad BglII (2526) 1. Lee HS, Spraggon G, Schultz PG, Wang F J Am Chem Soc (2009). 2. Yong TS, Ahmand I, Yin JA, Schultz PG J Mol Biol (2010). 11

12 Segmental labeling of membrane proteins using Sortase A. Sortase A: Staphylococcus aureus transpeptidase. Reduce NMR spectral complexity. Generate post-translationally modified proteins. CXCR1 Gai Sortase A Nanodiscs Various CXCR1 constructs Segmental labeling is used to specifically label a protein segment with NMR active nuclei, and as a result it reduces NMR spectral complexity, providing more flexibility in sample preparations and facilitate structural studies of multi-domain membrane proteins. 12